Method and apparatus for culturing hydrogen bacterium

Abstract

The present invention pertains to a method for culturing a bacterium belonging to the genus Hydrogenophilus in a culture solution while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein the risk of gas explosion caused by mixing the oxygen gas and the hydrogen gas is easily avoided by supplying the oxygen gas to the culture solution without previously mixing the oxygen gas with the hydrogen gas, whereby the bacterium belonging to the genus Hydrogenophilus can easily be cultured.

Description

Method and apparatus for culturing hydrogen bacteria

[0001] The present invention relates to a culturing method and apparatus capable of safely culturing hydrogen bacteria.

[0002] The Paris Agreement adopted in 2015 stipulates the rapid reduction of greenhouse gas emissions worldwide. In accordance with this, Japan aims to reduce greenhouse gas emissions such as carbon dioxide and methane by 26% compared to 2013 by 2030.

[0003] As a technology for fixing and effectively using carbon dioxide, attention has been paid to the technology of producing useful substances using microorganisms that utilize carbon dioxide. Hydrogen bacteria are chemosynthetic autotrophic bacteria that can grow using hydrogen as an energy source and carbon dioxide as a carbon source. Biomass and chemical products obtained by culturing hydrogen bacteria autotrophically have attracted attention as sustainable substances because carbon dioxide is the raw material.

[0004] Here, with the increase in the world population, the demand for food is increasing, and there is concern that food shortages will become a serious problem in the future. Among them, due to the limitations of the current livestock production system, there is concern that the balance between meat production and demand will be disrupted, and microbial protein is expected as a sustainable alternative protein that does not depend on livestock production. Therefore, the biomass obtained by the growth of hydrogen bacteria is being considered for use as an alternative protein in food and feed.

[0005] Also, most of the world's chemical production depends on petroleum raw materials, and there are problems such as an increase in greenhouse gas emissions. The method of producing organic substances by the original metabolism of hydrogen bacteria or genetic recombination of hydrogen bacteria has attracted attention as a method that does not depend on petroleum because carbon dioxide is used as the raw material. Also, there is high expectation for producing useful substances from carbon dioxide using hydrogen bacteria in order to reduce carbon dioxide, which is one of the greenhouse gases.

[0006] However, the technology for culturing hydrogen bacteria on a large scale has not been sufficiently developed. Hydrogen bacteria are aerobic and utilize oxygen as an electron acceptor. Hydrogen bacteria grow using the energy generated by the oxidation of hydrogen and carbon dioxide as a carbon source, so cultivation requires the supply of hydrogen, oxygen, and carbon dioxide. Conventionally, a method of supplying a mixed gas of hydrogen, oxygen, and carbon dioxide to the culture vessel has been adopted. This is because hydrogen bacteria belong to a group of bacteria that, although aerobic, exhibit the highest sensitivity to oxygen (Non-Patent Literature 1), and also because molecular oxygen strongly inhibits the growth of hydrogen bacteria in the early stages of growth, i.e., when the bacterial concentration is low (Non-Patent Literature 2). If oxygen gas, i.e., molecular oxygen, is supplied directly to the culture medium, hydrogen bacteria are exposed to high concentrations of oxygen gas, inhibiting their growth. Therefore, conventionally, when supplying oxygen during the cultivation of hydrogen bacteria, it has been supplied not as molecular oxygen, but as a mixed gas of hydrogen and carbon dioxide.

[0007] In the gas mixture used for culturing hydrogen-producing bacteria, the volume ratio of hydrogen:oxygen:carbon dioxide is usually 8:1:1 or 7:1:1, which is optimal for the growth efficiency of hydrogen-producing bacteria. The lower limit concentrations at which a mixture of hydrogen, oxygen, and carbon dioxide will explode are 4 v / v% for hydrogen and 6 v / v% for oxygen, and using a gas mixture in these ratios carries a risk of explosion. Therefore, it is necessary to establish a safe cultivation process.

[0008] For example, in the apparatus described in Non-Patent Documents 3-5, as a safety measure, a closed-system hydrogen gas culture apparatus is used to prevent the mixed gas from being released into the atmosphere. A gas chamber is attached to the culture vessel, and the gas supplied to the culture vessel is circulated between the culture vessel and the gas chamber, maintaining the oxygen concentration in that space below the explosion limit of the mixed gas. In addition, oxygen gas is supplied to the culture vessel via a separate line as needed. Furthermore, explosion-proof pumps and electrical systems are used for circulating or delivering the gas, and the generation of electrostatic sparks is prevented by using oil-free treated stainless steel pipes in the gas circulation line. Because the apparatus in Non-Patent Documents 3-5 has a gas chamber with a capacity of approximately 10 times that of the culture vessel, the apparatus becomes very large when used for industrial-scale cultivation. In addition, the cost is high because various equipment for explosion prevention is required.

[0009] E Wilde, et al., Antonie Van Leeuwenhoek, 1982, 48:131-43E Goto, et al., Agr. Biol. Chem., 1977, 41:521-5T Komada, et al., Agr. Biol. Chem., 1975, 39:77-82K Tanaka, et al., Biotechnol Bioeng. 1995, 45:268-75.K Tanaka, et al., Bioengineering 2023, 10, 1304.

[0010] The object of this invention is to provide a simple method and apparatus for safely culturing hydrogen bacteria.

[0011] The inventors have discovered that among hydrogen bacteria, bacteria of the genus Hydrogenophilus can grow well even when oxygen gas is supplied to the culture medium alone from the early stages of cultivation, i.e., when molecular oxygen is supplied.

[0012] The present invention has been completed based on the above findings and provides the following [1] to

[0015] . [1] A method for culturing Hydrogenophilus bacteria in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein the oxygen gas is supplied to the culture medium without being pre-mixed with the hydrogen gas. [2] The method according to [1], wherein a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas are supplied to the culture medium individually; a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas are supplied to the culture medium individually; or oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas are supplied to the culture medium individually. [3] The method according to [1] or [2], wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied to the culture medium in a volume ratio of 0.1 to 20: 1: 0.01 to 10. [4] The method according to any one of [1] to [3], wherein the total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas is 0.01 to 60 L / hour per L of culture medium. [5] The method according to any one of [1] to [4], wherein the hydrogen bacterium is Hydrogenophilus thermoreolus. [6] A method for producing a target substance by culturing Hydrogenophilus bacteria in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein the oxygen gas is supplied to the culture medium without being pre-mixed with the hydrogen gas. [7] The method according to [6], wherein a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas are supplied to the culture medium individually; a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas are supplied to the culture medium individually; or oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas are supplied to the culture medium individually. [8] The method according to [6] or [7], wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied to the culture medium in a volume ratio of 0.1 to 20: 1: 0.01 to 10. [9] The method according to any one of [6] to [8], wherein the total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas is 0.01 to 60 L / hour per L of culture medium.

[0010] The method according to any one of [6] to [9], wherein the hydrogen bacteria is Hydrogenophilus thermoreolus.

[0011] A culture apparatus for Hydrogenophilus bacteria comprising a culture vessel for holding a culture medium and a gas supply device for supplying hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium, wherein the gas supply device supplies oxygen gas to the culture medium without pre-mixing it with hydrogen gas.

[0012] The apparatus according to

[0011] , wherein the gas supply device either supplies a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas to the culture medium individually; supplies a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas to the culture medium individually; or supplies a gas containing oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas to the culture medium individually.

[0013] The apparatus according to

[0011] or

[0012] , wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium in a volume ratio of 0.1 to 20:1:0.01 to 10.

[0014] The apparatus according to any one of

[0011] to

[0013] , wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium such that the total supply rate of these gases is 0.01 to 60 L / hour per L of culture medium.

[0015] The apparatus according to any one of

[0011] to

[0014] , wherein the hydrogen bacterium is Hydrogenophilus thermortheorus.

[0013] Generally, hydrogen bacteria are known to have their growth inhibited when they come into contact with molecular oxygen, and this inhibition is particularly pronounced in the early stages of culture. For this reason, hydrogen bacteria have conventionally been cultured while supplying a mixed gas of hydrogen, oxygen, and carbon dioxide to the culture vessel. However, depending on the gas mixing ratio, this method carries the risk of explosion in the gas piping or reservoir. For this reason, a culture apparatus equipped with a large and complex explosion-proof closed-type gas-phase circulation system, such as the apparatus described in Non-Patent Documents 3-5, is required. Furthermore, there is the problem that it is not always possible to adopt the optimal gas mixing ratio for the growth of hydrogen bacteria.

[0014] The present invention solves these problems by supplying oxygen gas to the culture medium without pre-mixing it with hydrogen gas when culturing Hydrogenophilus bacteria. Therefore, the risk of explosion that occurs when a mixed gas containing oxygen gas and hydrogen gas is supplied to the culture vessel through piping or a reservoir can be avoided. In the method and apparatus of the present invention, some gas that is not consumed by the bacteria during cultivation may come out of the culture medium into the space inside the culture vessel, but usually only an amount that does not pose a risk of explosion comes out. Furthermore, the method and apparatus of the present invention only increases the number of lines for supplying gas to the culture vessel compared to the case in which a mixed gas is supplied to the culture vessel, and the apparatus does not become larger or more complex. Moreover, because there is no risk of explosion, the supply ratio of each gas can be easily set to a ratio suitable for the growth of the bacteria being cultured. Also, because there is no risk of explosion, a sufficient amount of gas can be supplied so that the bacterial cells can be cultured at high density. In this way, the present invention makes it possible to efficiently culture hydrogen bacteria at high density without requiring a large or complex culture apparatus and without the risk of explosion.

[0015] This is a schematic diagram of the culture apparatus used in the example to culture bacteria of the genus Hydrogenophilus.

[0016] The present invention will be described in detail below. (1) Method for culturing Hydrogenophilus bacteria and method for producing the target substance The present invention is a method for culturing Hydrogenophilus bacteria in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein the oxygen gas is supplied to the culture medium without being mixed with the hydrogen gas beforehand.

[0017] Examples of bacteria belonging to the genus Hydrogenophilus include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidance, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038. Bacteria of the genus Hydrogenophilus can be easily isolated from almost anywhere on Earth. Among them, Hydrogenophilus thermortheorus is preferred because it has a top-level growth rate and, consequently, carbon fixation capacity as a carbon-fixing microorganism. A preferred strain of Hydrogenophilus thermortheorus is strain TH-1 (NBRC 14978). Hydrogenophilus thermortheorus strain TH-1 (NBRC 14978) exhibits the highest growth rate among carbon-fixing microorganisms [Agricultural and Biological Chemistry, 41, 685-690 (1977)] (doubles in one hour). Hydrogenophilus thermortheorus strain NBRC 14978 is internationally deposited under the Budapest Convention and is publicly available. Hydrogenophilus bacteria may be bacteria isolated from nature, or bacteria that have been genetically modified from bacteria isolated from nature. For example, genetically modified strains can be created by constructing artificial metabolic pathways through genetic manipulation to confer new substance production capabilities.

[0018] When supplying each gas to the culture medium, oxygen gas and hydrogen gas are supplied independently. "Supplying independently" means supplying them separately without mixing. For example, a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas may be supplied to the culture medium individually; a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas may be supplied to the culture medium individually; or a gas containing oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas may be supplied to the culture medium individually. Each supplied gas may contain different gases to the extent that it does not inhibit the growth of Hydrogenophilus bacteria or the production of the target substance. A gas containing hydrogen gas, a gas containing oxygen gas, a gas containing carbon dioxide gas, a mixed gas containing oxygen gas and carbon dioxide gas, and a mixed gas containing hydrogen gas and carbon dioxide gas may be a gas consisting of hydrogen gas, a gas consisting of oxygen gas, a gas consisting of carbon dioxide gas, a mixed gas consisting of oxygen gas and carbon dioxide gas, and a mixed gas consisting of hydrogen gas and carbon dioxide gas, respectively. In this case, it is assumed that each gas contains an unavoidable amount of different gases.

[0019] The volume ratio (hydrogen:oxygen:carbon dioxide) of hydrogen gas, oxygen gas, and carbon dioxide gas supplied to the culture medium can be 0.1 to 20: 1: 0.01 to 10, and more specifically, 1 to 20: 1: 0.1 to 5, and more specifically, 1 to 10: 1: 0.1 to 3. Within this range, bacterial growth is good and the target substance can be produced efficiently. The volume ratio of the supplied gases may be constant during cultivation or may fluctuate.

[0020] In this invention, the culture medium can be stirred while culturing. Furthermore, the gas can be supplied continuously or intermittently.

[0021] The total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas can be 0.01 to 60 L / hour, 0.05 to 30 L / hour, or 0.1 to 10 L / hour per liter of culture medium. The gas supply rate may be constant or variable during cultivation. By adjusting the supply rate of each gas to match the material balance shown in the literature "Kodama. Petroleum and Microorganisms. 1997, 18: 32-41" according to the growth of the microbial cells, the growth of the bacteria will be good, the target substance will be produced efficiently, and gas waste will be minimized. For example, the supply rate of hydrogen gas can be 0.001 to 30 L / hour, 0.01 to 10 L / hour, or 0.05 to 5 L / hour per liter of culture medium. The oxygen gas supply rate can be 0.0001 to 10 L / hour, 0.0005 to 5 L / hour, or 0.01 to 1 L / hour per liter of culture medium. The carbon dioxide gas supply rate can be 0.0001 to 10 L / hour, 0.0005 to 5 L / hour, or 0.01 to 1 L / hour per liter of culture medium.

[0022] When supplying each gas into the culture medium in the culture vessel, it is sufficient to supply or blow the gas into the culture medium from the gas supply port at the end of the air supply piping that constitutes the gas supply line. The gas can be supplied as is or as fine bubbles. In either case, it is desirable that the supply ports for the oxygen gas and the hydrogen gas be separated, but they may be in contact.

[0023] The amount of culture medium should be sufficient to allow for adequate stirring by the stirring blades attached to the culture vessel, but it can be, for example, 0.1 to 250,000 L, 0.1 to 25,000 L, 0.5 to 2,500 L, 0.5 to 250 L, 1 to 25 L, or 1 to 2.5 L.

[0024] Since bacteria of the genus Hydrogenophilus can grow using hydrogen as an energy source and carbon dioxide as their sole carbon source, carbon dioxide can be efficiently fixed by culturing them using substantially only carbon dioxide as the carbon source (especially using only carbon dioxide). Therefore, in the method of the present invention, it is preferable to use an inorganic culture medium that does not contain carbon sources such as organic matter or carbonates, and to cultivate them using substantially only carbon dioxide as the carbon source (especially using only carbon dioxide as the carbon source). In the present invention, "using carbon dioxide as substantially the sole carbon source" includes cases where an unavoidable amount of other carbon sources are mixed in.

[0025] The pH of the culture medium is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within this range, bacterial growth and the solubility of each gas in the culture medium are high, resulting in good bacterial growth efficiency and substance production efficiency.

[0026] The culture temperature is preferably 35 to 55°C, more preferably 37 to 52°C, and most preferably 50 to 52°C. Within this range, bacterial growth is good, and the target substance can be produced efficiently.

[0027] The method for culturing Hydrogenophilus bacteria of the present invention described above can also be a method for producing a target substance when the target substance is to be produced by the Hydrogenophilus bacteria. The target substance is, for example, an organic compound. In addition to the culturing step, this production method may include a step of recovering the target substance from the culture medium.

[0028] (2) Culture apparatus for Hydrogenophilus bacteria The culture apparatus for Hydrogenophilus bacteria of the present invention comprises a culture vessel for holding a culture medium and a gas supply device for supplying hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium, wherein the gas supply device supplies oxygen gas to the culture medium without pre-mixing it with hydrogen gas. This apparatus is for carrying out the culture method for Hydrogenophilus bacteria of the present invention as described above.

[0029] The gas supply device is a device that supplies oxygen gas and hydrogen gas independently. The gas supply device may, for example, supply hydrogen gas, oxygen gas, and carbon dioxide gas individually to the culture medium; supply hydrogen gas and a mixed gas containing oxygen and carbon dioxide gas individually to the culture vessel; or supply oxygen gas and a mixed gas containing hydrogen and carbon dioxide gas individually to the culture medium. Each supplied gas may contain different gases to the extent that it does not inhibit the growth of Hydrogenophilus bacteria or the production of the desired substance. The hydrogen gas, oxygen gas, carbon dioxide gas, mixed gas containing oxygen and carbon dioxide gas, and mixed gas containing hydrogen and carbon dioxide gas may be, respectively, a gas consisting of hydrogen gas, a gas consisting of oxygen gas, a gas consisting of carbon dioxide gas, a mixed gas consisting of oxygen and carbon dioxide gas, and a mixed gas consisting of hydrogen and carbon dioxide gas. In this case, each gas may contain an unavoidable amount of different gases.

[0030] The gas supply system comprises a gas tank or a gas generator, and a gas supply line connecting them to the culture medium. The gas tank may be a high-pressure gas tank, and the gas generator may be, for example, a device that generates gas by vaporizing liquefied gas.

[0031] The gas supply line may include a supply pipe that delivers gas from a gas tank or gas generator to the culture medium, and a gas reservoir that stores gas before supplying it to the culture medium. A sparger may be provided at the gas supply port at the end of the supply pipe to make the gas supplied to the culture medium into fine bubbles. A gas supply line may be provided for each gas that is supplied individually. It is desirable that the supply ports for oxygen-containing gas and hydrogen-containing gas be separated, but they may be in contact. Even if they are in contact, the gas supplied to the culture medium will diffuse quickly, so it will not explode in the culture medium.

[0032] A regulator for adjusting the gas supply rate or gas supply amount may be attached to the gas tank or gas generator, gas supply piping, or reservoir. The volume ratio of hydrogen gas, oxygen gas, and carbon dioxide gas supplied to the culture vessel is the same as that of the culture method of the present invention described above. The total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas, and the supply rates of each gas, are also the same as those of the culture method of the present invention.

[0033] The capacity of the culture vessel can be determined according to the purpose of cultivation, but for example, it can be 1 to 500,000 L, 1 to 50,000 L, 1 to 5,000 L, 1 to 500 L, 1 to 50 L, or 1 to 5 L. The culture vessel may also be equipped with a stirrer for agitating the culture medium. The culture vessel may be equipped with a gas outlet, which may be connected to an exhaust pipe for gas discharge as needed. The culture vessel may be equipped with a temperature controller for adjusting the culture medium to the desired temperature. The temperature controller should be one that adjusts the culture medium temperature to the temperature described in the method of the present invention.

[0034] If the apparatus of the present invention is for continuous culture, it is sufficient to have a device for supplying new culture medium to the culture vessel and a device for draining old culture medium from the culture vessel. The device for supplying new culture medium to the culture vessel may include a culture medium tank, a supply pipe connecting the culture medium tank and the culture vessel, and a pump for delivering the culture medium, with the supply pipe being connected to the supply port of the culture vessel. The device for draining the culture medium from the culture vessel may include a drain pipe connected to the drain port of the culture vessel and a pump for delivering the culture medium. The device for supplying new culture medium to the culture vessel and the device for draining the culture medium from the culture vessel may work together to adjust the amount of culture medium in the culture vessel to the amount described in the method of the present invention.

[0035] This culture apparatus is for culturing bacteria of the genus Hydrogenophilus, and the bacteria of the genus Hydrogenophilus are as described in the present invention method.

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these. Culture apparatus A culture apparatus schematicly shown in Figure 1 was used. This culture apparatus comprises a culture vessel 1 and a gas supply device 2. The culture vessel 1 has an exhaust port 1a on its top surface and is equipped with a stirrer 3 for stirring the culture medium. The gas supply device has a hydrogen gas tank, an oxygen gas tank, and a carbon dioxide gas tank 2a, and three gas supply lines 2b. Each of the three gas supply lines 2b is equipped with an air supply pipe 2b1 extending from the gas tank into the culture vessel, and a gas pressure regulator 2b2 and a gas flow meter 2b3 are provided on the air supply pipe 2b1. The capacity of the culture vessel is 2L. Regarding the gas supply ports of the air supply pipe 2b1, the distance between adjacent ends of the oxygen gas supply port and the hydrogen gas supply port is 10cm. Although not shown, the culture vessel has an intake port and an outlet port for the culture medium and is equipped with a temperature controller for adjusting the temperature of the culture medium.

[0037] The culture apparatus shown in Figure 1 was used for culturing bacteria of the genus Hydrogenophilus. 1 L of liquid medium [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4・7H2O 0.014 g, MgSO4・7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4・7H2O 28 mg, CuSO4・5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4・5H2O 4.0 mg, CoCl2・6H2O 4.0 mg dissolved in 1 L of distilled water (pH 7.0)] was placed in the culture tank. Hydrogenophilus thermoreolus TH-1 (NBRC 14978) strain was inoculated into the culture medium and incubated at 52°C for 8 hours with stirring using an impeller (1,000 rpm). Hydrogen gas, oxygen gas, and carbon dioxide gas were supplied to the culture medium through separate gas lines. Hydrogen gas was supplied at a rate of 100 mL / hour for the first 2 hours of incubation, 150 mL / hour from 2 to 4 hours, 250 mL / hour from 4 to 6 hours, and 1,000 mL / hour from 6 to 8 hours. Oxygen gas was supplied at a rate of 30 mL / hour for the first 2 hours of incubation, 50 mL / hour from 2 to 4 hours, 75 mL / hour from 4 to 6 hours, and 300 mL / hour from 6 to 8 hours. Carbon dioxide gas was supplied at a rate of 25 mL / hour for the first 2 hours of incubation, 40 mL / hour from 2 to 4 hours, 60 mL / hour from 4 to 6 hours, and 250 mL / hour from 6 to 8 hours. The cells of Hydrogenophilus thermoreolus TH-1 strain proliferated approximately 200-fold in 8 hours of culture. No gas explosions occurred before, during, or after culturing.

[0038] The culture method and apparatus of the present invention can easily avoid the explosion risk associated with mixed oxygen and hydrogen gases, and can culture Hydrogenophilus bacteria at high density, making it highly valuable for industrial applications.

Claims

1. A method for culturing Hydrogenophilus bacteria in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein the oxygen gas is supplied to the culture medium without being pre-mixed with the hydrogen gas.

2. The method according to claim 1, wherein a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas are supplied to the culture medium individually; a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas are supplied to the culture medium individually; or oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas are supplied to the culture medium individually.

3. The method according to claim 1 or 2, wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied to the culture medium in a volume ratio of 0.1 to 20:1:0.01 to 10.

4. The method according to any one of claims 1 to 3, wherein the total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas is 0.01 to 60 L / hour per L of culture medium.

5. The method according to any one of claims 1 to 4, wherein the hydrogen bacterium is Hydrogenophilus thermorteolus.

6. A method for producing a target substance by culturing bacteria of the genus Hydrogenophilus in a culture medium while supplying hydrogen gas, oxygen gas, and carbon dioxide gas, wherein the oxygen gas is supplied to the culture medium without being pre-mixed with the hydrogen gas.

7. The method according to claim 6, wherein a gas containing hydrogen gas, a gas containing oxygen gas, and a gas containing carbon dioxide gas are supplied to the culture medium individually; a gas containing hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas are supplied to the culture medium individually; or oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas are supplied to the culture medium individually.

8. The method according to claim 6 or 7, wherein hydrogen gas, oxygen gas, and carbon dioxide gas are supplied to the culture medium in a volume ratio of 0.1 to 20:1:0.01 to 10.

9. The method according to any one of claims 6 to 8, wherein the total supply rate of hydrogen gas, oxygen gas, and carbon dioxide gas is 0.01 to 60 L / hour per L of culture medium.

10. The method according to any one of claims 6 to 9, wherein the hydrogen bacterium is Hydrogenophilus thermorteolus.

11. A culture apparatus for Hydrogenophilus bacteria comprising a culture vessel for holding a culture medium and a gas supply device for supplying hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium, wherein the gas supply device supplies oxygen gas to the culture medium without pre-mixing it with hydrogen gas.

12. The apparatus according to claim 11, wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium individually; supplies hydrogen gas and a mixed gas containing oxygen gas and carbon dioxide gas to the culture medium individually; or supplies oxygen gas and a mixed gas containing hydrogen gas and carbon dioxide gas to the culture medium individually.

13. The apparatus according to claim 11 or 12, wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium in a volume ratio of 0.1 to 20:1:0.01 to 10.

14. The apparatus according to any one of claims 11 to 13, wherein the gas supply device supplies hydrogen gas, oxygen gas, and carbon dioxide gas to the culture medium such that the total supply rate of these gases is 0.01 to 60 L / hour per L of culture medium.

15. The apparatus according to any one of claims 11 to 14, wherein the hydrogen bacterium is Hydrogenophilus thermortheorus.

Images